The research program is focused on the detailed mechanisms underlying the initiation of innate immune responses by the receptors and the ensuing signal transduction pathways. [unreadable] [unreadable] The innate immune system is the first line of defense against infection. It also initiates and directs the proper function of the adaptive immune response, the other branch of the immune system. The potency of the innate immune system has been harnessed as vaccination adjuvants for over eighty years. Nonetheless, the underlying mechanisms of action only started to be delineated about ten years ago, when a major family of the innate immune receptors (the TLRs) was identified. It is now appreciated that both TLR-dependent and TLR-independent innate immune activation regulate adaptive immune responses through various vaccine adjuvants. In fact, many ligands for the innate immune receptors are now under development as candidate vaccine adjuvants. Among exciting progress in the innate immune filed, the recent crystal structures of the human TLR3 extracellular domain have provided a framework upon which further investigation of the innate immune recognition can be conducted. However, without structures of these receptors in complex with their ligands, further progress in the field regarding the molecular mechanisms underlying innate immune recognition remain very limited. [unreadable] [unreadable] Our program addresses the above issues by integrating biochemical studies with extensive structural analysis of membrane bound (such as the Toll-like receptors) and cytoplasmic (such as RIG-I, NALP3 and ZBP1) receptors, either in complex with their ligands or downstream adapters/effector molecules. A critical feature of these innate immune receptors is that they distinguish among various classes of pathogenic molecules while retaining their capacity for responsiveness to a large number of related structures within a given biochemical class. How the binding domains of the innate receptors achieve such broad reactivity at the atomic level is one of the key issues this project addresses. Such information could be used to guide the development of new therapeutics that can either enhance or limit immune activation involving these receptors. Ligand binding by these receptors in turn initiates intracellular signaling cascades that ultimately lead to innate cellular responses that help fight infection and guide the adaptive immune responses. The project aims to decipher this signaling network through studying protein-protein interactions, using X-ray crystallography in conjunction with other biophysical and biochemical techniques. The ultimate goal is to not only delineate the mechanisms of innate immune responses at atomic details and contribute to our general understanding of signal transduction, but also to lay a foundation for future clinical exploitation of the innate immune system for human benefit, such as the development of more effective vaccine adjuvants.[unreadable] [unreadable] Progress has been achieved in the following areas during the year:[unreadable] [unreadable] 1). By taking advantage of the signal peptides from highly secreted proteins, we have been able to express recombinant extracellular domains of TLR5 and TLR7/8 as fusion proteins in HEK293 (human embryonic kidney) cells and CHO (chinese hamster ovary) cells. Both of the cell lines possess mutations in their glycosylation machinery such that the N-linked glycans of the secreted proteins are susceptible to endo-glycosidase degradation. This has been shown to be crucial in obtaining homogeneous glyco-protein samples for crystallization studies. We have yet been able to detect secreted expression of TLR9 extracellular domains, a known difficult target. We are utilizing different signal sequences to test additional TLR9 recombinant clones. In the meantime, we are initiating a collaboration with Eicke Latz of the University of Massachusetts Medical School on the conformational changes of TLR9 induced by microbial and synthetic CpG DNAs. The Latz group has been able to obtain TLR9 using different mammalian expression systems so we anticipate this will be a very productive collaboration for both parties. [unreadable] [unreadable] 2). In collaboration with Jenny Ting from the University of North Carolina, we have successfully expressed and purified an amino-terminal fragment of NLR family member NALP12/Monarch-1. In contrast to most members of the NLR family, Monarch-1 attenuates the inflammatory responses by suppressing the activation of NF-&#954;B upon TLR stimulation. Therefore, Monarch-1 serves as a crucial link between the TLR and NLR family members that may be important in preventing pro-inflammatory responses from developing into auto-inflammatory disorders. We have achieved high level expression of Monarch-1 in both insect cells and mammalian cells (HEK 293). Crystallization of the purified protein is underway. [unreadable] [unreadable] 3). By engineering bacteria expression vectors with multiple expression and purification tags, as well as utilizing metabolic regulation of gene expression from T7 promotors, we have achieved soluble expression of the TIR, CARD and Pyrin domains. These include the TIR domains from human MyD88, TIRAP, TLR1, TLR2, TLR4, TLR9 and TICAM, as well as those from mouse MyD88 and mouse TICAM; the CARD domains from human RIG-I, MDA5 and IPS-1; and the Pyrin domains from human NALP1, NALP3, ASC and POP1/ASC2. Most of these expressed proteins have been purified and the crystallization experiments are underway. The above domains were selected based on known signaling partners, which will allow us to study the protein-protein interactions among them and crystallize the interacting domain pairs, for example, the TIR domains of MyD88 and TIRAP, the CARD domains of RIG-I and IPS-1, and the Pyrin domains of NALP3 and ASC. [unreadable] [unreadable] 4). We have developed insights into the dimerization behavior of the TIR domains, particularly that from MyD88, a central adaptor molecule for the majority of the TLRs. The dimerization of MyD88 has been shown to be a crucial event in signal transduction. Using purified recombinant MyD88 TIR domains, we have demonstrated that the dimerization may proceed in two stages: the first is through non-covalent interactions, perhaps using the BB loop, a critical element of TIR-TIR interaction identified by mutagenesis. This is then followed by the formation of covalent disulfide bonds, most likely through the Cysteine near the BB loop. In collaboration with Peter Schuck of the Protein Biophysics Resource section at the DBEPS, ORS of NIH, we are using analytical ultracentrifugation to gain further insights into the thermodynamics of the dimerization. [unreadable] [unreadable] 5). Using highly purified samples, we have demonstrated that there is weak if at all interactions among the CARD domains of IPS-1 and RIG-I or MDA5 in vitro, consistent with the hypothesis that additional mitochondria membrane proximal proteins may be the necessary components of the signaling complexes of RIG-I or MDA5. In collaboration with Jenny Ting at the University of North Carolina, whose lab has identified another mitochondria protein involved in the RIG-I/MDA5 signaling pathway, we will study these signaling complexes using biochemical and structural methods. [unreadable] [unreadable] 6). Similar to the CARD domains, we have also shown that there is very weak association between the Pyrin domains of the central adaptor molecule ASC and its partner POP1/ASC2 or NALP3. In addition to the Pyrin domain, ASC also contains a CARD domain that was proposed to bind the CARD domain of caspase-1 in the inflammasome complex. We are testing the hypothesis that inclusion of the CARD domain of ASC may facilitate the binding of ASC and its partner proteins involving Pyrin domains, which is conducive to the formation of inflammasomes. [unreadable] [unreadable] 7). We have recently established a collaboration with Stefan Rothenburg at NICHD to study a new cytosolic DNA sensor ZBP1/DAI.